The ultimate goal of the project is developing a new method for non-invasive clinical imaging of activity of specific enzymes. Enzymes of the matrix metalloproteinase (MMP) family are known to play a significant role in progression of cancer, atherosclerosis, and other inflammation related disease. The ability to identify a specific enzyme and localize its activity using 3D imaging would help physicians to better understand disease progression and response to treatment. The method proposed here combines a smart molecular agent and a 3D photoacoustic imaging (PAI) technique. PAI provides a way to embed optical information on ultrasound imaging. The images feature high resolution and high penetration depth along with the specificity and contrast of optical imaging. The specific implementation of PAI that our team proposes relies on probing excitation lifetime. The technique is termed photoacoustic lifetime imaging (PALI). We propose designing a molecular probe that consists of two methylene blue (MB) chromophore molecules linked by a peptide chain that confines the chromophores to a dimer configuration. The peptide link is designed to be cleaved upon interaction with MMP-2 enzyme. This would result in breaking the MB dimer and releasing two MB monomers. The two states of the probe, MB dimer vs. free MB monomers, exhibit a very drastic difference in excitation lifetime. MB monomers have a stable triplet excited state that has a natural lifetime of 70 ms. The triplet state is very effectively quenched in a dimer configuration resulting in a very short lifetime of less than 0.040 ms. This extreme difference in lifetime between the probe in its initial state and its activated state provie an effective contrast mechanism for PALI imaging. Moreover, the activated state of the probe is clearly distinct from any other tissue component because of the extremely long excitation lifetime of the chromophores. This mechanism would therefore generate high image contrast for functional imaging of enzyme activity. The research plan entails the following steps: 1. Probe Synthesis: Synthesize and test a molecular probe consisting of MB pair tethered to a peptide sequence that can be cleaved by MMP-2. 2. Probe Characterization: Measure sensitivity and specificity to MMP-2. 3. Testing imaging features such as penetration depth, sensitivity, and contrast in tissue-mimicking phantoms. 4. Testing the new imaging system in vivo in mice models of prostate cancer. The project will lay the groundwork for a translational research that would focus on implementing the technique in clinical applications such as cancer screening, treatment planning, and treatment monitoring.